Proc. Natd Acad. Sci. USA Vol. 78, No. 11, pp. 6888-6892, November 1981 Botany

Morphology of a novel cyanobacterium and characterization of light-harvesting complexes from it: Implications for evolution (//cell wall//prokaryotic evolution) THOMAS A. KURSAR*, HEWSON SwIFTt, AND RANDALL S. ALBERTEt tBarnes Laboratory, Department ofBiology, and *Department of Biophysics and Theoretical Biology, University of Chicago, Chicago, Illinois 60637 Contributed by Hewson Swift, July 2, 1981

ABSTRACT The morphology of the marine cyanobacterium After examining the in vivo spectral properties of several of DC-2 and two light-harvesting complexes from it have been char- the recently discovered species ofcyanobacteria, it came to our acterized. DC-2 has an outer cell wall sheath not previously ob- attention that one of the PE-containing types termed DC-2 served, the purified phycoerythrin shows many unusual proper- showed some rather unusual features. Further study revealed ties that distinguish it from all characterized to that this species possesses novel PE, phycobilisomes, and outer date, and isolated phycobilisomes have a single absorption band cell wall sheath; these characteristics suggest that it should be at 640 nm in the - region of the spec- trum. On the basis of these observations we suggest that DC-2, placed in a new phylogenetic branch for the . rather than being a member of the group, should be placed in its own taxonomic group. In addition, the particular MATERIALS AND METHODS properties of the isolated phycoerythrin suggest that it may be An axenic representative of an early stage in the evolution of the phyco- isolate of Synechococcus sp., clone DC-2, obtained erythrins. These observations are ofspecial interest in light ofthe from R. R. L. Guillard, was grown in f/2 enriched seawater contribution DC-2 and related cyanobacteria may make to global medium (9) at 20°C with aeration. Cells were disrupted in a primary productivity. French pressure cell and cellular debris and membranes were removed by centrifugation. The supernatant was fractionated Several species of blue-green- and red-pigmented marine cy- with ammonium sulfate to purify PE. The 20-40% saturated anobacteria that not only are abundant in the world's oceans but ammonium sulfate cuts were combined and dialyzed against 10 also may be responsible for a significant fraction ofprimary pro- mM sodium phosphate (pH 6.8) containing 0.2 M sodium chlo- ductivity have been discovered recently (1, 2). The red-pig- ride at 5°C. The dialyzed sample was loaded on hydroxylapatite mented cells have the virtue that they are readily distinguished and eluted with increasing concentrations ofsodium phosphate from most other phytoplankton species by their principal in vivo (pH 6.8). The fractions having the highest Au3/A2w were fluorescence emission in the orange (560-580 nm), which un- pooled and rechromatographed as before. This PE fraction was doubtedly arises from the presence ofthe red pigment-protein, passed through a Sephadex G-200 column equilibrated with 50 phycoerythrin (PE). In some cyanobacteria and most , mM sodium phosphate (pH 7.0). The main fraction was eluted PE serves as the major light-harvesting pigment for photosyn- with 50 mM sodium phosphate (pH 7.0), collected and rechro- thesis and is found in association with the other major phycobili- matographed on G-200. The A543/A2w ratio was 8.2 after both pigments, phycocyanin and allophycocyanin. In vivo these phy- the second and third passes through G-200. The final yield of cobilipigments are aggregated into macromolecular arrays that PE was 31%. All chromatographic procedures were conducted form discrete organelles called phycobilisomes and are attached at 20°C. -like particles were prepared from the to the a-containing photosynthetic lamellae (3). cells as described (10). All spectral characterizations were con- Such an organization allows for excitation energy transfer from ducted at 20°C on either an Aminco DW-2 dual beam spectro- the shortest-wavelength-absorbing pigment, PE, to phycocy- photometer or an SPF-500 corrected spectrum fluorometer. A anin and allophycocyanin and ultimately to the Cary 60 spectropolarimeter was provided by the Department residing in the photochemical reaction centers ofphotosystems of Chemistry, University of Chicago. The concentration of the I and II (3-5). Therefore, the phycobilipigments serve two im- (PUB) and (PEB) chromo- portant functions in photosynthesis: (i) they increase photon phores were measured in 8 M urea (pH 2.0) containing 10 mM capture under light-limited conditions and (ii) they increase the 2-mercaptoethanol (11). spectral range of light energy available for photosynthesis by Cells were prepared for electron microscopy by fixing in 4% absorbing light where chlorophyll a absorbs weakly and where (wt/vol) glutaraldehyde in ultrafiltered sea water buffered to there is the greatest transmission oflight in the water column. pH 7.4 with 10 mM sodium cacodylate. The cells were postfixed Recent studies have demonstrated that the in 1% osmium tetroxide, embedded in Epon, and stained with have highly conserved NH2-terminal sequences (6-8) and that uranyl acetate and lead citrate. phycobilisome structure is also fairly conservative (3). The dual requirements for efficient energy transfer from the phycobili- RESULTS AND DISCUSSION pigments to chlorophyll a and for assembly of a phycobilisome seem to impose strong constraints on the nature ofthe phycobil- With the exception of the outer sheath, the cells of DC-2 re- ipigments and their in vivo macromolecular organization. sembled the related genera Anabaena (12) and Microcystis (13) in structure. Cell profiles were 0.6-1.2 Im in width (average The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: PE, phycoerythrin; PUB, phycourobilin; PEB, phy- nmnt" in accordance with 18 U. S. C. §1734 solely to indicate this fact. coerythrobilin; CD, circular dichroism. 6888 Downloaded by guest on September 29, 2021 Botany: Kursar et aL Proc. NatL Acad. Sci. USA 78 (1981) 6889 0.8), and were up to 4.0 Aum in length (Fig. la). Cell division energy within the pigment-protein to longer wavelength (flu- involved constriction and binary fission as in related genera. A orescing) chromophores at the 565-nm absorption band (20-23). distinctive feature of DC-2 is its outer sheath, composed of a In isolated PE these chromophores will fluoresce, whereas in series ofparallel ridges spaced about 300 A apart, serrate in cross vivo the excitation energy is transferred to an adjacent light- section, branched at ends ofthe cell, and running parallel to its harvesting pigment-protein. long axis (Fig. lb). Sheath components in Anacystis (14) and The 543-nm absorption peak of DC-2 PE assigned to PE is Anabaena (15) lack this complex orientation. Ridges were at- symmetrical and relatively narrow; the bandwidths at halfmax- tached at their bases to a trilaminar extracellular membrane, imum of DC-2 PE and C-PE are 1060 cm-' and 1880 cm-', similar to that described for other cyanobacteria (16). In some respectively. Denaturation ofPEs with detergents, heat, urea, cells (e.g., as in Fig. 1c), cytoplasmic lamellae were closely ap- sulfhydryl reagents, etc. results in the loss of the long-wave- pressed to the inner surface of the cell membrane, producing length absorption band and the appearance of a nearly sym- a multilayered cell margin. metrical maximum at about 540-550 nm (24-29). This repre- The absorption spectrum of the purified PE of DC-2 has sents the absorption maximum of a protein-bound PEB maxima at 543, 497, 375, and 304 nm (Fig. 2). The chromo- molecule for which there is a minimal protein-chromophore phores of PE include PUB and PEB, which are linear tetra- interaction. The 565-nm absorption bands of the PEs are be- pyrroles related to the bile pigment . The 497-nm lieved to be due to either protein-chromophore or chromo- band is assigned to PUB and the 375- and 543-nm bands are phore-chromophore interactions (26). Therefore native DC-2 assigned to PEB. The ratio of PEB to PUB in the purified PE PE has an absorption maximum similar to that ofdenatured PEs was determined to be 4:1 (see Materials and Methods). Most and the distinct "fluorescing" PEB found in all other PEs may PEs previously described, including the C-, B-, and R- spec- be (i) absent, (ii) blue-shifted 10-20 nm, or (iii) found only in troscopic types, have a markedly asymmetric absorption spec- the fully aggregated complex. These observations suggest that trum with a maximum at 560-565 nm and a second maximum the PEBs ofDC-2 PE have electronic and vibrational states that or shoulder at 545 nm (7, 17); for example, the R-PE of Gra- are only weakly perturbed by the protein. The fluorescence cilaria (Fig. 2). Two exceptions are the PE ofOscillatoria (Tri- emission spectrum of DC-2 PE has a maximum at 563 nm and chodesmium) thiebauti and PE-I of the cryptomonads, which a shoulder at 605 nm. The main emission band is also blue- have a shoulder rather than a maximum at 550-560 nm (18,19). shifted with respect to other PEs, all of which have emission From an analysis ofthe polarization offluorescence otherwork- peaks at 570-578 nm (Fig. 2; ref. 7). ers have concluded that 545-nm band of C-, B-, or R-PE rep- The visible circular dichroism (CD) spectrum ofDC-2 PE has resents "sensitizing" PEB chromophores that transfer excitation several unique properties (Fig. 3). First, the negative Cotton

a b

c

FIG. 1. Electron micrographs of chroococcalean cyanobacterium DC-2. (a) Characteristic cells from a centrifuged pellet. (x40,000.) (b) Portion of two cells showing the sheath cut in cross section (right) and in tangential section (left). (x80,000.) (c) Part of another cell showing polyhedral bodies, photosynthetic lamellae, small cytoplasmic vesicles, and the cell wall-sheath complex. (x 120,000.) Downloaded by guest on September 29, 2021 U090ACO Botany: Kursar et aL Proc. NatL Acad. Sci. USA 78 (1981)

N

2-20

X

-401

-60

500 600 Wavelength, nm Wavelength, nm FIG. 2. Room-temperature absorption (-) and fluorescence FIG. 3. CD spectra of DC-2 PE in 50 mM sodium phosphate, pH emission (----) spectra of purified PEs from DC-2 (a) and Gracilaria 7.0 (-), and 8 M urea/10 mM 2-mercaptoethanol, pH 2.0 (----), with tikvahiae (b). The spectra of DC-2 PE were measured in 50 mM sodium scanning rates of 1.5 and 5 nm/min, respectively. The concentration phosphate (pH 7.0) and those of Gracilaria PE were measured in 100 of PEB was 9.56 pM and the estimated protomer concentration was mM sodium phosphate (pH 5.5). Both emission spectra were measured 2.39 uM. The vertical bars indicate root mean square noise levels. with samples having an absorbance less than 0.05 at the 540- to 570- nm maxima. The excitation wavelength was 500 um andthe excitation that either the PEB is in an and emission bandpasses were 15.0 and 2.0 nm, respectively. Emission is, trapped asymmetric conformation spectra were corrected on a quantum basis. (possibly helical) or the PEB excited state is interacting with dipoles or chromophores of the protein (31). Hence the large decrease in ellipticity upon denaturation ofPseudanabaena PE effect at 542 nm is not found in C-, B-, or R-PE, all of which demonstrates that the native optical activity arises from have a positive Cottoneffect at this wavelength (27, 29). Second, PEB-protein interaction. The intense CD ofthe native complex the ellipticity ofthe 497-nm PUB absorption is negligible (Fig. has been explicitly interpreted as indicating that the PEB as- 3), whereas both B- and R-PE have positive Cotton effects at sumes a helical conformation (29). A similar explanation has 497 nm (27). been given to account for the intense CD ofthe biliverdin-hu- In comparison, native PE from Pseudanabaena has a positive man serum albumin complexes and the lack of detectable CD Cotton effect at 546 nm that is about 4.5 times more intense than ofthefree biliverdin (Table 1; refs. 30-32). Incontrast, denatur- that of DC-2 PE. Upon denaturation ofthe.Pseudanabaena PE ation of DC-2 PE results -in relatively small changes in the vis- in urea, this CD band loses about 85% ofits intensity (Table 1). ible CD (Fig. 2; Table 1). The weak positive band is lost. The Because the "intrinsic" optical activity ofPEB is probably quite main band, shifted to 572 nm, is still negative and the integrated small, the observed optical activity is likely to be "extrinsic;" ellipticities ofthe negative Cotton effects ofthe native and dena-

Table 1. Visible CD spectra of native and denatured forms of two PEs and of free and bound biliverdin -Awna,, [0] x 10-4, Sample Buffer nm deg cm2/dmol Molar units Ref. PE of DC-2 50 mM NaPi, pH 7.0 542 -24.3 4 PEB (1 protomer)* This paper PE of DC-2 8 M urea, pH 2.0/10 mM 572 -15.1 4 PEB (1 protomer)* This paper 2-mercaptoethanol PE of Pseudanabaena W 1173 25 mM NaPi, pH 7.0 546 + 109.2 5 PEB (1 protomer) 29 PE of Pseudanabaena W 1173 8 M urea, pH 7.0 525 +16.6 5 PEB (1 protomer) 29 580 +13.1 Biliverdin-human serum albumin 20 mM Tris-HCl, pH 650 -12 1 biliverdin 30 1:1 complex 7.4/0.1 M NaCl Free biliverdin 20 mM Tris HCl, pH 650 Not 30 7.4/0.1 M NaCl detectable * The-concentration of PEB was determined. The ratio of 4 PEB per protomer is an estimate. Downloaded by guest on September 29, 2021 Botany: Kursar et al Proc. NatL Acad. Sci. USA 78 (1981) 6891

tured proteins are approximately equal. The native protein ap- may have a single absorption band in the phycocyanin-allophy- parently derives very little CD intensity from the pro- cocyanin region ofthe spectrum. All cyanobacteriaand red algae tein-chromophore interaction, which may indicate that the as- are believed to contain phycocyanin, which has an absorption sociation of the PUB and PEB chromophores with the protein maximum at 612-630 nm, and allophycocyanin, with a maxi- is weak. mum at 650 nm. Within the intact isolated phycobilisome, ex- The unusual spectral properties ofisolated PE are probably citation energy is transferred from PE to phycocyanin to allo- very similar to the in vivo properties. First, the PE in the phy- phycocyanin and emitted as fluorescence with a quantum yield cobilisome has nearly equivalent absorption properties (Fig. 4). of about 0.7 (3). Two requirements must be met in order to Secondly, by ammonium sulfate fractionation one can isolate a obtain such efficient energy transfer: the chromophores must spectrally pure PE from DC-2 in less than 10 hours that has have an appropriate spatial organization, and the fluorescence nearly identical absorption, fluorescence, and CD spectra, the emission spectrum ofthe donor pigment-protein must overlap major difference being that the fresh material has a 70% increase with the absorption spectrum of the acceptor complex (35). in the magnitude of the ellipticity at 543 nm. Crude phycobilisome preparations from DC-2 were analyzed It is likely that during the evolution of the phycobiliproteins on linear sucrose gradients, which resolved three colored bands the binding of a buin chromophore to a protein was a first step. (Table 2). The smallest particles (band I) had their main fluo- This idea is supported by studies of the fluorescence yield of rescence emission at 563 nm due to free PE. The intermediate- model compounds. Free bilins or pyrromethenes (the latter are sized particles (band II) had a major fluorescence emission band analogues of the two central rings of the bilin chromophore) at 650 nm and a minor band at 563-567 nm. The largest par- have a quantum yield offluorescence that is less than 0.001 and ticles, the phycobilisomes (band III), represented only 8% of may have two paths for radiationless deactivation of a singlet the total PE recovered (Table 2). The low yield of phycobili- state: intramolecular proton exchange between the pyrrole ni- somes appears to be due to their unusual instability. They had trogens and twisting modes of the methine-ring bonds (33). a minor fluorescence emission maximum at 563-567 nm and Bilins bound to protein or pyrromethenes complexed with bo- a major band at 674 nm, which are comparable to the values ron or bromide can have fluorescence quantum yields that are reported for the phycobilisomes isolated from other organisms 102 or 103 times higher than the yield ofthe free chromophores (3). The isolated phycobilisomes have an absorption maximum (33). It is possible that highly fluorescent bilin conformers can at 543 nm due to PE and a second absorption maximum at 640 be selectively stabilized by both covalent and noncovalent pro- nm that is yet to be assigned (Fig. 4). DC-2 appears to be unique tein-chromophore interactions. The assembly of such a pig- among the cyanobacteria and red algae in having only a single ment-protein complex suggests itself as the first step in the symmetrical absorption band in the 600- to 650-nm region (Am. utilization ofthe bilins for photosynthesis. Further, attachment = 640 nm; Fig. 4 Inset). A pigment-protein complex with an of chromophores to a protein matrix permits the formation of absorption maximum at 634 nm is also present in the band II ordered aggregates that do not show the fluorescence quench- particles (Fig. 4). The fluorescence ofpurified DC-2 PE, with ing common to randomly aggregated chromophores (4, 34). The a maximum at 563 nm and a shoulder at 620 nm, only weakly unique absorption, fluorescence, and CD properties ofthe PE overlaps with the 640-nm absorption band, suggesting a poor from DC-2 suggest that it may be representative of an early coupling in the transfer ofexcitation energy between these two stage in the evolution of the PEs. The more common C-, B-, states. The small 572-nm shoulder in the absorption spectrum and R-PEs, which have 565-nm absorption bands along with ofDC-2 phycobilisomes (Fig. 4, arrow) may, however, indicate distinct "sensitizing" and "fluorescing" PEB chromophores, the presence of long-wavelength "fluorescing" PEB chromo- long-wavelength fluorescence, and more intense optical activ- phores that are found only in the fully assembled phycobili- ity, probably evolved at a later time. some. The 640-nm absorption band of the phycobilisomes can The phycobilisomes of DC-2 are also unusual because they be interpreted (i) as a phycocyanin-allophycocyanin complex with unusual spectral properties, or (ii) as a single pigment-protein that replaces the phycocyanin-allophycocyanin pair, in which case it may have additional absorption bands(s) to the blue such as PC637 ofAgmanellum quadruplicatum or PC I ofHemiselmis virescens (36, 37). It is not especially difficult to accept the sec- ond interpretation that allophycocyanin is absent, in view ofthe fact that the cryptomonads lack allophycocyanin and are able to transfer excitation energy to reaction centers (37). Chloro- phyll c, which may function as a bridging pigment in crypto- monads (37), is absentfrom DC-2 (unpublished data). Excitation of the PE of intact phycobilisomes results in a minor fluores- cence emission peak at 563 nm due to PE and a major emission peak at 674 nm (Fig. 3). The low level of PE emission suggests that energy transfer within the phycobilisome is very efficient

Table 2. Color, relative yield, and fluorescence characteristics of DC-2 sucrose gradient fractions 300 400 500 600 700 nm Relative Fluorescence Wavelength, Band Color yield,* % maxima, nm FIG. 4. Absorption ( )and fluorescence emission (--)spectra I Orange 39 563 of phycobilisomes of DC-2-in 0.75 M NaKPi, pH 7.2, at 20°C. The ex- II Red-purple 53 563-567, 650 ciainwvlnta7nm adtJ xi m Deep purple 8 563-567, 674 passes were 20.0 and 2.0 nm, respectively. Other conditions were as described for Fig. 2. (Inset) Expanded absorption spectra of phycobili- * The relative yield was determined as the percent of the total PE somes (-) and band II particles (----). recovered. Downloaded by guest on September 29, 2021 A660092 Botany: Kursar et aL Proc. Nat Acad. Sci. USA 78 (1981)

and that the overlap of PE fluorescence with the 640-nm pig- 8. Glazer, A. N., Apell, G. S., Hixson, C. S., Bryant, D. A., Ri- ment-protein is adequate. The quantum yield offluorescence, mon, S. & Brown, D. M. (1976) Proc. Natl. Acad. Sci. USA 73, though, remains to be measured. In contrast, intact DC-2 cells 428-431. 9. McLachlan, J. (1973) in Handbook ofPhycological Methods, ed. show an unusually large in vivo fluorescence emission ofFw3/ Stein, J. R. (Cambridge University Press, Cambridge, England), F6w equal to 0.86-1.32, depending on light intensity during pp. 25-51. growth. Possibly this fluorescence arises from the peculiar 10. Gantt, E., Lipschultz, C. A., Grabowski, J. & Zimmerman, B. K. properties of the light-harvesting system of DC-2 (e.g., aggre- (1979) Plant Physiol 63, 615-620. gation state, chromophore environment, etc.) or is the result 11. Glazer, A. N. & Hixson, C. S. (1977)J. Biol, Chem. 252, 32-42. of PE that is not bound to the phycobilisomes. 12. Gantt, E. & Conti, S. F. (1969) J. Bacteriol 97, 1486-1493. 13. Lauritis, J. A., Vigil, E. L., Sherman, L; & Swift, H. (1975) J. Ultrastruct. Res. 53, 331-334. CONCLUSIONS 14. Jost, M. (1965) Arch. Mikrobiol 50, 211-245. By several criteria, Synechococcus clone DC-2 is a novel cy- 15. Echlin, P. (1964) Protoplasma 58, 439-457. 16. Leak, L. V. (1967) J. Ultrastruct. Res. 21, 61-74. anobacterium. These include (i) the unique absorption, fluo- 17. O'Carra, P. & O'hEocha, C. (1976) in Chemistry and Biochem- rescence, and CD spectra ofits PE, (ii) isolated phycobilisomes istry of Plant Pigments, ed. Goodwin, T. W. (Academic, Lon- that have a single 640-nm absorption peak in the 600- to 650-nm don), pp. 328-376. region, (iii) the large in vivo fluorescence of PE from whole 18. Fujita, Y. & Shimura, S. (1974) Plant Cell Physiol 15, 939-942. cells, and (iv) the unique outer sheath morphology. Further 19. Morschel, E. & Wehrmeyer, W. (1977) Arch. MicrobioL 113, analyses of the light-harvesting system and the phylogenetic 83-89. 20. Dale, R. E. & Teale, F. W. J. (1970) Photochem. Photobiol 14, relationships ofthis organism may be essential for a full under- 163-173. standing of the phycobiliproteins and of evolution among the 21. Vernotte, C. (1971) Photochem. Photobiol 14, 163-173. cyanobacteria. Further, the novel features ofthis cyanobacter- 22. MacColl, R. & Berns, D. S. (1978) Photochem. Photobiol 27, ium and its great abundance in global marine systems demands 243-249. examination of its photosynthetic properties, adaptive physi- 23. Zickendraht-Wendelstadt, B., Friedrich, J. & Rudiger, W. (1980) ology, and role in primary productivity. Photochem. Photobiot 31, 367-376. 24. O'hEocha, C. & O'Carra, P. (1961) J. Am. Chem. Soc. 83, 1091-1093. We thank Dr. R. R. L. Guillard for bringing the existence of DC-2 25. Jones, R. F. & Fujimori, E. (1961) Physiol Plant. 14, 253-259. to our attention and for providing axenic cultures and Drs. G. Holz- 26. Macdowall, F. D. H., Bednar, T. & Rosenberg, A. (1968) Proc. warth, M. Makinen, and G. Fleming for helpful discussions. The initial Nat! Acad. Sci. USA 59, 1356-1363. examinations reported here were conducted as part ofthe Experimental 27. Pecci, J. & Fujimori, E. (1969) Biochim. Biophys. Acta 188, Marine Botany Program (1979) at the Marine Biological Laboratory, 230-236. Woods Hole, MA, supported in part by National Science Foundation 28. Talarico, L. & Kosovel, V. (1978) Photosynthetica 12, 369-374. from National Science Foun- 29. Langer, E., Lehner, H., Ridiger, W. & Zickendralt-Wendel- Grant PCM 79-06638. Additional support stadt, B. (1980) Z. Naturforsch. 35c, 367-375. dation Grant PCM 78-10535 (to R.S.A.) and National Institutes of 30. Blauer, G. & Zvilichovsky, B. (1973) Isr. J. Chem. 11, 435-443. Health Grant GM23944 (to R.S.A.) is acknowledged. R.S.A. was the 31. Blauer, G. (1974) Struct. Bonding (Berlin) 18, 69-129. recipient of an Andrew W. Mellon Foundation Fellowship. 32. Blauer, G. & Wagniere, G. (1975) J. Am. Chem. Soc. 97, 1949-1954. 1. Waterbury, J. B., Watson, S. W., Guillard, R, R. L. & Brand, L. 33. Holzwarth, A. R., Lehner, H. Braslavsky, S. E. & Schaffner, K. I. (1979) Nature (London) 277, 293-294. (1978) Justus Liebigs Ann. Chem. 1978, 2002-2017. 2. Johnson, P. W. & Sieburth, J. (1979) LimnoL Oceanogr. 24, 34. Beddard, G. S. & Porter, G. (1967) Nature (London) 260, 928-935. 366-367. 3. Gantt, E. (1980) Int. Rev. Cytot 66, 45-80. 35. Forster, T. (1965) in Modern Quantum Chemistry, ed. Sinan- 4. Porter, G. (1978) Proc. R. Soc. London Ser. A 362, 281-303. oglu, 0. (Academic, New York), Part 3, pp. 93-137. 5. Grabowski, J. & Gantt, E. (1978) Photochem. PhotobioL 28, 36. Gray, B. H., Cosner, J. & Gantt, E. (1976) Photochem. PhotobioL 47-54. 24, 299-302. 6. MacColl, R. & Berns, D. S. (1979) Trends Biochem. Sci. 4, 37. Gantt, E. (1979) in Biochemistry and Physiology ofthe Protozoa, 44-47. eds. Levandowsky, G. M. & Hutner, S. H. (Academic, New 7. Glazer, A. N. (1977) Mole CelL Biochem. 18, 125-140. York), 2nd Ed., Vol. 1, pp. 121-137. Downloaded by guest on September 29, 2021